Diagnostic assays based upon the absorbance (chromogenic, colorimetric), light scattering (turbidimetric or nephelometric), and emission of analytes in biological systems are known in the art. In each of these assays, an analyte in a biological system of interest is determined, i.e., its concentration in the biological system of interest, by reacting the analyte of the system with specific assay reagents and monitoring the changes in the either the chromogenic, turbidimetric or emissive properties of the reaction medium. These methods measure the change in the optical properties, that is, the transmittive or emissive properties of an assay solution resulting from the presence of a particular ligand in the assay solution.
For example, in a spectrophotometric assay, the analyte in the sample to be determined and a reagent system specific for the analyte, produces a detectable change in the transmittive properties of the assay solution. The transmittive properties change according to the amount of light absorbed or scattered by an assay solution within a particular wavelength band when a beam of light of known intensity is passed through the assay solution.
Colorimetric assays are a type of spectrophotometric assay in which the change in the transmittive properties, e.g., absorbance, of the solution results in a change in the light absorption of the assay solution. The change in the absorption of the assay solution results, directly or indirectly, from the interaction of the analyte to be determined and the reagent system specific for the analyte. The change in absorbance of the assay solution is related to the concentration of the analyte in the assay solution. Colorimetric assays utilize a chromogenic reagent system capable of interacting in an assay solution with the particular ligand of interest.
Turbidimetric or nephelometric assays are other spectrophometric assays that measure the scattering of light by the assay solution rather than the changes in color. Turbidimetric assays determine the amount of light scattered or blocked by particulate matter as light passes through the assay solution. In these assays, the analyte of interest interacts with a reagent system to form a suspension of particles in the assay solution. As a beam of light having a known intensity is passed through an assay solution, the suspension of particles formed by the interaction of the analyte and reagent system scatters the incident light thereby reducing the intensity of the light transmitted through the assay solution. These assays measure the decrease in the intensity of the light transmitted through an assay solution. The decrease is related to the amount of incident light that is scattered or blocked by the suspension of particles and depends upon the number of particles present and the cross-sectional area of such particles.
Nephelometric assays are similar to turbidimetric assays in that the analyte of interest interacts with a reagent system specific for the analyte to form a suspension of particles in the assay solution. Nephelometric assays measure the amount of incident light scattered by the suspension of particles. Unlike a turbidimetric assay wherein the intensity of the light transmitted through the assay solution is measured, in a nephelometric assay the scattered light is measured at an angle to the light incident to the assay solution. Therefore, in a nephelometric assay the change in the transmittive properties refers to the difference in intensities of light incident to the assay solution and light scattered at an angle to the incident light.
Fluorometric assays typically measure a detectable change in the fluorescent properties of the assay solution. The assay medium is excited with monochromatic light of a wavelength within the excitation wavelength band of the fluorescer. The change in the fluorescent properties of the assay solution is delivered by measuring the intensity of the emitted light at a wavelength within the emission wavelength band of the fluorescent molecule. The intensity of the emitted fluorescent light is related to the concentration of the analyte.
Fluorescent assays are based upon the principle that when a fluorophore is irradiated with light of the appropriate wavelength, the fluorescent intensity of the light emitted by a reaction medium is directly proportional to both the concentration of fluorophore in the reaction medium and the intensity of excitation light impinging the medium. Fluorescent analyzers generally relate changes in the fluorescent intensity of the reaction medium to changes in the concentration of emitting moieties in that medium, by holding the intensity of the excitation light constant. Reagents for fluorescent assays are designed so that the concentration of emitting fluorophores in the reaction medium is changed in proportion to the concentration of the analyte in the sample of the medium.
In general, the analytical instruments designed to measure change in the optical properties of the reaction medium are limited to changes in one type of signal, namely, the signal associated with the absorbent, light scattering and fluorescent properties of the medium. For example, an instrument designed for use in a fluorometric assay generally is not applicable for chromogenic or turbidimetric determinations, without extensive modification of the optics of the instrument to accommodate fluorescence. In the event reaction media that absorb or scatter light also exhibits fluorescence, changes in absorption or scattering may be detected by measurement of change in fluorescence in an instrument designed to detect fluorescence, without the capability of directly detecting chromogenic or turbidimetric changes in the media.
U.S. Pat. No. 4,495,293 describes a method for determining a ligand in a sample suspected of containing the ligand, wherein the method comprises combining to form an assay solution: the sample, an effective amount of a fluorescer; and an effective amount of a reagent system which in the presence of the ligand to be determined is capable of providing a change in the transmittive properties of the assay solution within a wavelength band that overlaps the excitation and/or emission wavelength band of the fluorescer; irradiating the assay solution with light having a wavelength within the excitation wavelength band of the fluorescer; and then measuring the intensity of the fluorescence emitted by the assay solution as a measure of the concentration of the ligand in the sample. In these methods, the fluorescer is added to the assay solution and thus, great care must be taken that there will be no chemical or immunological reaction between the fluorescer and any other component of the assay, e.g., the ligand or reagent system. The fluorescer may also be dependent on the pH range of the reaction system.
It would be useful to have a system for measuring chromogenic or turbidimetric changes in an assay solution using a fluorometric system. It would especially be useful to have a fluorometric system where the fluoroscer or fluorophore does not come in contact with the reagent system.
U.S. Pat. No. 5,173,434 to Morris et al. describes a process to detect or determine the concentration of a substance that directly or indirectly functions to change the light transmissive characteristics of a solution. The method involves providing a light beam forming a transmission light path; providing a solution including said substance in said light path; the improvement comprising positioning a fluorophore, in a chemically inert light transparent matrix, to intersect said light path; and detecting the change in fluorophore emission to determine the presence or concentration of said substance. The path length of the excitation light through the test solution in Morris is typically 8 mm. Morris does not describe test devices for performing the method described therein which enable the detection of substances in very small sample volumes.
It is desirable to minimize the amount of sample that is necessary for performing the assay. For example, a limited supply of sample may be available for testing, e.g., on neonates. In addition, small samples require fewer reagents, reducing the costs associated therewith. However, the sensitivity of the assay may be compromised when using very small sample sizes. Thus, it would be desirable to have a device that enables use of very small sample sizes, but which does not sacrifice assay sensitivity.
Furthermore, diagnostic assays typically require the mixing of the sample of interest with the components of the reagent systems prior to performing the assay. This may require the reagents to be prepared before running the assay. If the reagents have a short shelf life, the reagent may have to be prepared frequently, which may be costly and which may introduce additional errors into the system. Similarly, having to prepare reagents decreases the time efficiency of the assay. It would therefore be desirable to have a device which contains some or all of the reagents for the assay and which can be used in an automated system for performing diagnostic assays. Such devices would increase the time and cost efficiency of diagnostic assays.